Fungicidal composition comprising a salicylate and a pyrazole



United States Patent Ofiice 3,072,525 Patented Jan. 8, 1963 The present invention relates to a highly effective fungicidal composition and more particularly to a fungicidal composition containing, as active ingredients, salicylic acid esters and other fungicidal agents.

It is one object of the present invention to provide a new and highly eifective fungicidal composition which comprises, in combination, salicylic acid esters and other antifungal agents and wherein such a combination produces a surprisingly high synergistic effect.

Another object of the present invention is to provide highly effective antifungal compositions for combatting fungi pathogenic to plants.

A further object of the present invention is to provide highly effective disinfectant compositions to be used for many purposes.

Other objects of the present invention and advantageous features thereof will become apparent as the description proceeds.

According to the present invention it has been found that the combination of salicylic acid esters with other antifungal compounds, such as salicylic acid anilide, esters of p-hydroxy benzoic acid, or, preferably, antifungal hydroxy phenyl pyrazole compounds, produces a noteworthy increase in antifungal activity which increase is due to a surprising synergistic effect.

According to Walter and Heilmeyen, Antibiotika- Fibel, published by Georg Thicme Verlag, Stuttgart, 1954, and Websters New International Dictionary, published by G. C. Merriam Co., Springfield, Mass, 2nd. ed., 1948 the term synergism is defined as the cooperative action of discrete agencies such that the total effect is greater than the sum of the two eifects taken independ ently. According to Marwyk, Zeitschrift fuer Hygiene, vol. 144, p. 477 (1958), who quotes from the literature, synergism is defined as any of the combined doses being smaller than its half minimum inhibiting dose.

While esters of salicylic acid per se' exert only a small antimycotic effect, their combination with the fungicidal compounds mentioned above yields compositions which even in high dilution, have a surprisingly high effect against a large number of fungi, especially against fungi affecting plants. dead organic matter, and animals.

Esters of salicylic acid which are useful in compositions according to the present invention are esters with low molecular aliphatic alcohols and glycols such as they are obtained by the addition of one or more moles of ethylene oxide to salicylic acid, as well as esters with phenols.

Preferred fungicidal hydroxy phenols pyrazole compounds are, for instance:

3-(2-hydroxy phenyl) pyrazole,

S-(Z-hydroxy-S-chloro phenyl) pyrazole,

3-(2-hydroxy-4-chloro phenyl) pyrazole,

3-(2-hydroxy-5-bromo phenyl) -5-methyl pyrazole, and

others.

Of fungicidal esters of p-hydroxy benzoic acid, especially the esters with lower aliphatic alcohols with an alkyl chain of l to 4 carbon atoms may be used.

The compositions according to the present invention have proved to be especially effective in combatting fungi pathogenic to plants such as Claviceps purpurea, Gnomania erythrostoma, Sphaceloma ampelinum, Coniothyrium diploa'iella, Phytium de Baryanum, Cenagium abietis, Cepha losporium acremonium, Hemispora stellata, Penicillium spinolosum, Penicillium notatum, and others.

Although the compositions according to the present invention are especially valuable against plant-pathogenic fungi, they are not limited thereto, but can be used in combating any kind of fungus infection or attack. They may, for instance, be used as disinfectants in swimming pools, shower baths, mining installations, for cleaning 7 wooden gratings, board-walks, as an additive to soap and detergents used for the disinfection of articles of daily use, such as linen, pottery, earthenware, porcelain articles, rubber shoes, hospital equipment as well as for the control of pathogenic germs in rooms. For the last mentioned purpose th composition may be incorporated into agents used for the maintenance and care of floors, walls,

etc, such as floor polishing waxes.

Due to their effectiveness against plant pathogenic fungi the compositions according to the present invention can also effectively be used against fungus diseases of grain, grapevine, cherry trees, firs, pines and others. They are, furthermore, useful for protecting wine barrels, wine cellars, food stocks, textiles, and the like against fungal attacks.

The high penetrating power of the new compositions is of special value in their use as disinfectants, preservatives, and fungicidal agents for combating plant-pathogenic fungi.

The present invention and the synergistic effect of the new combination are illustrated by the following comparative tests. Tables I and II show the results obtained by using various compositions on Trichophyton gypseum, and Tables III and IV show the results obtained by the action of a composition consisting of methyl salicylate and 3-(2-hydroxy-5-chloro phenyl) pyrazole on various plant-pathogenic fungi.

, The values given in Tables I and II show the minimum inhibitory concentration of each compound and the concentration of the combination of the antifungal agents which cause complete inhibition of growth. .The tables show that, when using less than 50% of the minimum inhibitory concentration of the salicylic acid ester and less than 50% of the minimum inhibitory concentration of the other fungicidal component, complete inhibition of growth of the test germ T richophyton gypseum is achieved.

TABLE I Test Tube Dilution Test [Observation time: 21 days] Complete Percent 7 inhibition of of the Test Test compound Nutrient growth in a minimum No. medium concentration inhibitory of concentration 1 Methyl salicylate B 3.000

2 3 (2 hydroxy 5 chloro B 200,000 100 phenyl) pyrazole.

3. Combination of methyl sa- B 10,000 33.3 licylate and 3-(2-hydroxy- '2, 000, 000 10. 0 fi-ehloro phenyl) pyrazole 4 Ethyl sallcylate A 4, 600 100 5 Salicylic acid auilide A 64.000 100 6"-.- Combination of ethyl salie- A 20, 480 22 ylate and salicylic acid 256,000 25 anilide 7 n-Propyl salicylate B 10,000 100 8...-.. Methyl ester of p-hydroxy B 2, 500 100 benzoic acid 9"--. Combination ofnpropyl sa- B 40,000 25 licylate and methyl ester 125,000 2 of p-hydroxy benzoic acid 10-..- n-propyl salicylate A 2, 400 100 11 Methyl ester of p-hydroxy A 2,000 100 benzoic acid TABLE I-Continued Test Tube Dilution Test-Continued Complete Percent inhibition of the Test Test compound Nutrient growth in a minimum No. medium concentration inhibitory of 1:- concentration 12.... Combination oin-propyl sa- A 20, 480 12 licylate and methyl ester 256, 000 0.8 of p-hydroxy benzoic acid 13.. n-butyl salicylate B 1,000 100 14...- Salicylic acid anilide B 50,000 100 15...- Combination of n-butyl sa- B 2,500 40 licylate and salicylic acid 1,000,000 anilide 16..-- -do B 5,000 20 500, 000

17..- n-Butyl salicylate A 800 100 Salicylic acid anilide A 64,000 100 19 Combination of n-butyl sa- A 10,240 8 licylate and salicylic acid 130,000 49 anilide 20...- Phenyl salicylate B 8,000 100 3 (2 hydroxy 5 chloro B 200,000 100 phenyl) pyrazole 22. Combination of phenyl sa- B 20,000 40 licylate and 3-(2-hydroxy- 1, 000, 000 20 5-chloro phenyl) pyrazole 23---- -do B 40,000 20 500,000 40 Nutrient medium A: Bacto-Sabouraud maltose agar (Diico.). Nutrient medium E: Standard I nutrient Do 25.0 parts, glucose 10.0 parts, glycerol 5.0 pa

As is evident from the tests of Table I, the fungicidal effect of the antifungal compounds used in said tests is increased considerably by a combination with salicylic acid esters regardless what nutrient medium is used. As a result of such a combination synergistic effects are uillon Merck 250 parts, agar rts, distilled water ad 1,000 cc.

achieved as they have not been observed heretofore.

The new active compositions according to the present invention are furthermore characterized by a remarkably stable intensity of activity under practical conditions of use, even in the presence of protein, as well as by a note- Ordinarily fungicidal agents worthy power of diffusion.

are substantially completely inactivated by solutions containing 5% of protein. In contrast thereto the active compositions according to the present invention retain their activity and their synergistic eifect in the presence of protein as is evident from the tests shown in the following Table II.

5 TABLE II Test Tube Dilution Test With the Addition of 5% of Bovine Serum [Test period: 21 days] 10 Complete Percent inhibition of the Test Test compound Nutrient of growth minirrum No. medium 1 in a conenin ibitory tration of concen- 1: tration 24 Methyl salieylate B 3,000 100 3 (2 hydroxy 5 chloro B 200, 000 100 phenyl) pyrazole.

26..-- Combination of methyl sali- B 10,000 33.3

cylate and 3-(2-hydroxy- 2,000,000 10 o-chloro phenyl) pyrazole.

27..-. n-Propyl salicylate 13 6,000 100 Methyl ester of p-hydroxy B 2, 500 100 benzoic acid.

29..-- Combination of n-propyl B 40,000 15 salicylate and methyl 125,000 2 ester of p-hydroxy benzoic acid.

25 aon-Butylsalicylate B 700 100 31..-. Salicylic acid anilide B 50. 000 100 32.... Combination of n-butyl B 2, 500 28 salicylate and salicylic 1,000,000 5 acid anilide.

33-.-. (lo B 5,000 14 500,000 10 34. Phenyl salicylatc B 8,000 100 35... 3 (2 hydroxy 5 chloro B 200, 000 100 phenyl) pyrazole.

30.". Combination of phenylsali- 20,000 cylatc and 3-(2-iydroxy- 1, 000. 000 20 5-el1loro phenyl) pyrazole.

1 See footnote 1, Table I.

The results of both Tables I and II prove that combinations of 3-(2-hydroxy-5-chloro phenyl) pyrazole with esters of salicylic acid are highly effective.

The following Tables III and IV show the effect of such combination against fungi which are pathogenic to plants and attack dead organic material. The test methods used are the same as described for Tables I and II.

TABLE III Test Tube Dilution Tests With Various Fungi [Observation period: 21 days] Corn plcte Percent of inhibition the mini- Tcst compound Test fungus Nutrient of growth in mm inmedium 1 aconcentrahihitory tion of l: concentration Methyl salicylate C'laviceps purpurca 0 3,000 3-(2-hydroxy-5-chloro phenyl) pyra ole Claviccps pumurca-.. 0 25,000 100 Combination of methyl salicylate and 3-(2-hydroxy-5- Claviceps purpurea C 10,000 33,3 chloro phenyl) pyrazole. 100,000 25 Methyl salicylate Gnomonia crz/throstomc D 3,000 100 3-(2-hydroxy-5-chlnro phenyl) pyrarole Gnomonia crythros oma D 25,000 100 Combination of methyl salicylute and 3(2-hydroxy-5- Gnomom'a crythrostoma D 5), 000 About 6 chloro phenyl) pyrazole. 00, 000 About 42 Methyl salicylate Sphaceloma ampelinum D 4, 000 100 3-(2-hydroxy-5-chloro phenyl) pyra olenn Sphaceluma ampelz um. D 25,000 100 Combination of methyl salicylate and 3-(2-hydroxy-5- Sphaceloma ampelmimn D 40,000 10 chloro phenyl) pyrazole. 000 About 42 Methyl salicylfite" C rium diplo iiella D 5,000 100 3-(2-hydroxy-5-chloro phenyl) pyrnzole Conioihyrium diplodiella D 25, 000 100 Combination of methyl salicylate and 3-(2-hydroxy-5- Coniothyrium iliplodiella D 50,000 10 chloro phenyl) pyrazolc. 00,000 About 42 Methyl salicylate Phi/Hum de hart/(mum- D 4,000 100 3-(2-hydroxy-5-chloro phenyl) nyrazole Phi/Ilium de b rl 11mm- D 10,000 100 Combination of methyl salicylate and 3-(2-hydroxy-5- Phytium de barzanmm D 10,000 40 chloro phenyl) pyrazole. 25,000 40 Methyl salicylate- C ,um abz' tis 0 4,000 100 3-(2-hydroXy-5-chloro phenyl) pyrazole Cenanpium abiet 0 10,000 100 Combination of methyl salicylate and 3-(2hydroxy-5- Cenangzum abiel 0 50,000 8 chloro phenyl) pyrazole. 50,000 20 TABLE III-Continued Test Tube Dilution Tests, Etc.--Continued Complete Percent of inhibition the mini- Test compound Test fungus Nutrient of growth in mum inmedium 1 a concentrahibitory tion of 1: concentration ethyl salicylate c'ephalosporium acremonium.- C ,000 100 3-(2-hydroxy-5-chloro phenyl) pyraz'ole Cephalospori'um acre'm0nium V C v 10, 000 100 Combination of methyl salicylate and 3-(2-hydroxy-5-chloro Cephalosporium acremm'u-m C 10, 000 phenyl) pyrazole. 25,000 40 Methyl salicylate Hemispora stellata o 5, 000 100 3-(2-hydroxy-5-chloro phenyl) pyrazole Hemispora stellata 0 25,000 100 Combination of methyl salicylate and 3-(2-l1ydroxy-5-chloro Hemispora stellatu C 12, 000 About 42 phenyl pyrazole. r 100, 000

Methylsalicylate Penicillium spi'nolosum A 2,000 100 3-(2-hyrlroxy-5-chloro phenyl) pyrazole Pe'nicillzjum spinolo um. A 25,000 100 Combination of methyl salicylate and 3-(2-hydroxy-5-chloro Pemcillzum spmolosum A 10,000 20 phenyl) pyrazole. 60,000 About 42 Methyl salicylate. P I ium notaium A 2, 000 100 3-(2-l1ydroxy-5-chloro-phenyl) pyrazole Pentczllqum notatum A 25,000 100 Combination of methyl salicylate and 3-( iydroxy-5-chloro Pemcillmm notatum A 10. 000 20 phenyl) pyrazole. 60,000 About 42 Nutrient medium A: Bacto-Sabouraud maltose agar (Difco). Nutrient medium B: Standard glucose 10.0 parts, glycerol 5.0 parts, distilled water a 0 cc. Nutrient medium C: Beer wort agar.

nutrient bouillon Merck 25.0 parts, agar 25.0 parts,

Nutrient medium D: Oat flake agar.

(b) Combinations of methyl salicylate with 1 3-( Z-hydroxy-S-chloro phenyl) pyrazole, (2) Methyl ester of p-hydroxy benzoic acid,

(3) Salicylic acid anilide.

Each of the four components were administered orally by way of a stomach sound in doses of 150 mg, 300 mg., 600 mg., 1200 mg., and 2400 mg, per kg. of body weight to 6 test anmials for each dose. Within the test period TABLE IV Test Tube Dilution Test With the Addition of 5% Bovine Serum [Observation period 21 days] Complete Percent of inhibition the mini- Tost compound Test fungus Nutrient of growth in mum inmedium 1 a ooncentrahibitory tion of l: concentration Methyl salicylate Trichophyton gypsaum w. B 3, 000 100 3-(2-11ydroxy-fi-cbloro-phenyl) pyrazole Tmhophyton gypseum B 200,000 100 Combination of methyl salicylate and 3-(2-hydroxy-5-chloro- Trz'chophyton gupseum B 10, 000 33. 3 phenyl) pyrazole. 2,000, 000 10 Methyl salicylate P trachciphila G 4, 000 100 3-(2-hydroxy-5-chloro phenyl) pyrazole Pseudopeztzu trachegphgla O 25, 000 100 Combination of methyl salicylatc and 3-(2-hydroxy-5-chloro Pseudopeztza tmchezphzla C 10, 000 40 phenyl) pyrazole. 100,000 25 Methyl salicylate Phytium de barz/anum D 3,000 100 3-(2-hydroxy-5-ehloro phenyl) pyrazole. Phyiium de barycmum D 10, 000 100 Combination of methyl salicylate and 3-(2-hydroxy Plug Hum de baryanum D 10,000 33. 3

phenyl) pyrazole. 25,000 40 Methyl salicylate Hemispom stellata--- C 4, 000 100 3-(2-hydroxy-5-chloro ph l) pyrazol Hemispora slellata- 0 25,000 100 Combination of methyl salieylate and 3-(2- Hemispara stellata- C 10, 000 40 phenyl )pyrazole. 100, 000 25 1 See footnote 1, Table III.

of 3 days no animal died when receiving doses up to 1200 mg./kg. When doses of 2400 mg./kg. were administered, only 3 of 6 test animals in the group treated with methyl salicylate died.-

Higher doses were not administered because it is unlikely that higher doses will be of any importance for all practical purposes. Thereby, it is also to be taken into account that, as a rule, compounds which are lethal to animals in a dose exceeding 1000 mg./ kg. of body weight, are considered to be substantially non-toxic.

When using combinations of methyl salicylate with each of the above'mentioned fungicidal compounds, doses of (a) 600 mg./kg. of body weight and V (b) 1200 mg./kg. of body weight were administered orally by means of an esophageal sound. The above amounts of 600 mg./kg. and 1200 mg./kg. of body weight consist of varying proportions of methyl salicylate and the other antifungal component, namely (1) 1 part of methyl salicylate and 9 parts of the other antifungal agent,

(2) 9 parts of methyl salicylate and 1 part of the other antifungal agent, or

(3) 5 parts of methyl salicylate and 5 parts of the other antifungal agent.

With combinations of methyl salicylate and 3-(2-hydroxy-S-chloro phenyl) pyrazole in the ratios and doses mentioned above none of the treated test animals died.

With combinations of methyl salicylate and the methyl ester of p-hydroxy benzoic acid 2 of 6 animals died when using a dose of 600 mg./kg. of body weight and compositions of the ratios 1:9 and 5:5. When using compositions of the ratio 9:1 in a dose of 1200 mg./kg. of body weight, 2 of 6 test animals died. All other test animals stayed alive. As a result of these tests it can be concluded that only with a relatively high dose of methyl salicylate in the combination with the' methyl ester of phydroxy benzoic acid some toxic effects are observed.

When using combinations of methyl salicylate with salicylic acid anilide, none of the test animals died within 3 days on administration of doses of 600 mg./kg. and 1200 mg./ kg. of body weight regardless what ratio of the two components was used.

According to these acute toxicity tests it can be concluded that the increase in fungicidal activity on combining salicylic acid esters with other fungicidal agents is not due to a corresponding increase in toxicity to warmblooded animals.

The following examples provide formulas of compositions according to the present invention without, however, being limited thereto.

EXAMPLE 1 Percent Solution:

3-(2-hydroxy-5-chloro phenyl) pyrazole 4-(N-phenyl-N-benzyl)-amino-l-methy1 piperidine lactate 1.0 Methyl salicylate 0.01 Polyoxy ethylene sorbitan monopalmitate known as Teen 40 10.0 Isopropanol 40.0 Water, ad 100.0 percent.

EXAMPLE 2 Ointment:

3-(2-hydroxy-5-brorno phenyl)-S-methyl pyrazole 3.0 4-(N-phenyl-N-benzyl)-amino-1-methy1 piperidine lactate 1.0 Methyl salicylate 0.05 Anthrasol 2.0 Polyoxy ethylene sorbitan mono-oleate known as Tween 80 15.0 Ethanol 10.0 White wax 5.0 Hardened peanut oil 31.0 Water, ad 100.0 percent.

EXAMPLE 3 Powder:

3-(2-hydroxy phenyl) pyrazole 3.0 Methyl salicylate 0.1 Silica of the Acrosil type 7.5 Emulsifying wax known as Lanette N (Na-ethyl sulfate 10%, ethyl stearyl alcohol 90%) 1.0 Product known as Cetiol V (natural oils of animal origin, esters of oleic acid with liquid fatty alcohols) 0.5 Talc, ad 100.0 percent.

EXAMPLE 4- Powder:

3-(2-hydroxy-4-chloro phenyl) pyrazole 2.0 Monoethyleneglycol salicylate 0.2 Silica of the Aerosil type 7.5

Emulsifying wax known as Lanette N (Na-ethyl sulfate 10%, ethyl stearyl alcohol 1.0 Product known as Cetiol V (natural oils of animal origin, esters of oleic acid with liquid fatty alcohols) 0.5 Talc, ad 100.0 percent.

'In the first two formulas 4-(N-phenyl-N-benzyl)- amino-l-methyl piperidine lactate is added as an agent allaying itching.

It is, of course, understood that many changes and variations in the salicylic acid ester employed, the antifungal agent used, the amounts and the proportions of salicylic acid ester and antifungal agent in compositions according to the present application, the mode of application, the composition of the antifungal preparations, and the like may be made by those skilled in the art in accordance with the principles set forth herein and in the claims annexed hereto.

As stated above, the preferred compositions according to the present invention are compositions containing salicylic acid esters and 3-(2-hydroxy phenyl) pyrazole compounds. Such compositions contain preferably about 3% of the pyrazole compound and 0.01% to 0.1% of the salicylic acid ester. Ordinarily, amounts of about 5% of the pyrazole compound and about 1% of the salicylic acid ester should not be exceeded in such preparations.

The preferred compositions according to the present invention which are composed of a salicylic acid ester and a hydroxy phenyl pyrazole, preferably a chloro hydroxy phenyl pyrazole, are also suitable for the treatment of fungus diseases of the skin of humans and animals. The synergistic effect of the'components of such compositions is demonstrated on a large number of fungi, especially on pathogenic skin fungi such as Triclzophyton schoenleinii, Trichophyton epz'lans, T richophyton gypseum, and the like as well as on Microsporum species, such as M icrosporum gypseum, Microsporum distortum, on Epidermophyton species, such as Epidermophyton floccosum and Epidermophyton interdigitale, on Turolopsis species, such as Turolopsis candida, on Blastomyces species, such as Blastomyces dermatidis and Blastomyces brasiliensis, on Sporotrichum species, such as Sporotrichum schenckii, on Aspergillus species, such as Aspergillus fumigatus and Aspergillus niger, and others.

Due to their broad spectrum of effectiveness a considerable number of fungus diseases of the skin have been successfully treated, for instance, dermatomycoses, such as epidermophytoses, favus, microsporoses, pityriasis, erythrasma, trichophytoses, as well as various forms of moniliasis and sporotrichosis.

The esters of salicylic acid have the highly advantageous property of readily penetrating the skin. They serve, at the same time, as solvents for the other fungicidal component, for instance, for hydroxy phenyl pyrazole and chloro hydroxy pyrazole. Due thereto, the compositions according to the present invention are especially suitable for the treatment of all skin fungus diseases, where a good penetration into the skin is of special importance.

Furthermore, compositions according to the present invention possess properties which render them especially suitable for practical application; such properties are, for instance, their low toxicity against warm-blooded animals.

I claim:

1. An antifungal composition comprising, as antifungal agents, a lower alkyl ester of salicylic acid and 3- (2-hydroxy-5-chloro phenyl) pyrazole.

2. An antifungal composition comprising, as antifungal agents, methyl salicylate and 3-(2-hyd1'oXy-5-chloro phenyl) pyrazole.

3. An antifungal composition comprising, as antifungal agents, between about 0.01% and about 1.0% of methyl salicylate and between about 0.05% and about 5.0% of 3-(2-hydroxy-5-chloro phenyl) pyrazole.

4. An antifungal composition comprising, as antifungal agents, between about 0.01% and about 1.0% of a lower alkyl ester of salicylic acid and between about 0.05%

and about 5.0% of 3-(2-hydr0Xy-5-chloro phenyl) pyrazole.

5. The process of inhibitnig fungal growth consisting in subjecting the fungus growth to the action of a composition comprising, as antifungal agents, methyl salicylate and 3-(2-hydroxy-5-chloro phenyl) pyrazole.

References Cited in the file of this patent UNITED STATES PATENTS 2,721,143 Kraft et a1. Oct. 18, 1955 2,764,614 Meyer Sept. 25, 1956 2,802,029 Schuler Aug. 6, 1957 OTHER REFERENCES Gross et al.: The Salicylates, Hillhouse Press, New

10 Haven (1948), pages 149-151.

15 Dept. of Agr. Bulletin ET-223, recd February 5, 1947,

pages 1-7.

Schuler: German application, Ser. No. Sch 10069, printed September 27, 1956 (K30h 9), 3 pages spec. 

1. AN ANTIGUNGAL COMPOSITION COMPRISING, AS ANTIFUNGAL AGENTS, A LOWER ALKYL ESTER OF SALICYLIC ACID AND 3(2-HYDROXY-5-CHLORO PHENYL) PYRAZOLE. 